The instant invention relates to tissue cultures and more particularly to an in vitro method and apparatus for growing tissue cells under conditions which more closely resemble cells grown in vivo.
Experiments wherein tissue cells are grown in in vitro tissue cultures have produced significant amounts of data relating to tissue development over the past 70 years. However, it has generally been found that the cells of specimens which are grown in static, in vitro environments develop as randomly oriented embryonic cells which lack the three-dimensional adult characteristics of cells grown in vivo. Recently, however, it has been found that by applying stimulation consisting of gradual stretching forces to tissue specimens grown in in vitro tissue cultures, the tissue cells thereof tend to align with the stretching forces and they therefore exhibit somewhat more mature characteristics. However, it has been found that even the cells of specimens which are exposed to stimulation consisting of gradual stretching forces fail to develop into adult-like, three-dimensional structures which are similar to those grown in vivo. It has also recently been found that by applying stimulation consisting of repetitive or reciprocating stretch and release activity to tissue specimens grown in in vitro tissue cultures, the cells thereof tend to align in perpendicular relation to the direction of the stretch and release activity, and still fail to develop into three-dimensional structures similar to those grown in vivo.
It has now been found that when in vitro culture cells are stimulated by applying a mechanical activity pattern thereto which more closely resembles the type of stimulation pattern which naturally occurs in vivo, it is possible to grow the cells of a tissue specimen into structures which more closely resemble tissue cells grown in vivo. Specifically, it has been found that by simultaneously applying both continuous, gradual stretch activity and periodic reciprocal stretch and release activity to a tissue specimen grown in an in vitro culture, it is possible to develop the cells of the tissue specimen into well formed, three-dimensional structures which are similar to those grown in vivo. In this connection, by continuously stretching a tissue specimen, the tissue specimen is subjected to the type of mechanical stimulation which normally results from animal growth in vivo; and by periodically repetitively stretching and releasing the tissue specimen, the tissue specimen is subjected to the type of stimulation to which in vivo tissue cells are exposed during animal movements.
In accordance with the above, the method of the subject invention comprises the steps of supporting a tissue specimen in the gross morphology found in vivo (aligned or unaligned as in vivo) with an expandable membrane so that the tissue specimen is expandable therewith, maintaining the tissue specimen in an extracellular matrix, such as a collagen matrix, maintaining the tissue specimen in a life support environment, periodically supplying fresh nutrients to the tissue specimen, and gradually stretching the membrane by at least approximately 0.5% over a 24 hr period while periodically reciprocally stretching and releasing the membrane by at least approximately 0.01% for a total of at least 60 seconds during the course of the same 24 hr period. The tissue specimen is preferably stretched and released by between 0.02% and 60% during the stretch and release activity periods, and the stretch and release activity periods preferably last at least approximately 5 seconds. The stretch and release activity periods are preferably separated by rest periods of at least approximately 5 seconds, and the tissue specimen is preferably gradually and continuously stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr. The membrane preferably has a pair of spaced, upwardly extending support walls thereon which are made of a biocompatible material and have opposed support surfaces thereon, and the tissue specimen is preferably allowed to attach to the support surfaces of the support walls, and it is stretched by moving the support walls apart.
When the method of the subject invention is applied to a tissue specimen comprising isolated cells requiring alignment, the tissue specimen is preferably applied to a resiliently expandable membrane which is adapted to permit attachment of the tissue specimen thereto, such as by preapplying a collagen coating thereto. Thereafter, the method is carried out by maintaining the tissue specimen in a life support environment, periodically feeding the tissue specimen, and maintaining the cells of the tissue specimen in an extracellular matrix sufficient to permit stretching of the tissue specimen without causing damage thereto. Then, while maintaining these conditions, the membrane is gradually stretched over a prolonged period of time until the cells are substantially aligned in the direction of stretch, and thereafter the membrane is gradually stretched by at least approximately 0.5% over the course of a 24 hr period while it is periodically stretched and released by at least approximately 0.01% for a total of at least 60 seconds during the course of the same 24 hr period. The tissue specimen is preferably stretched and released by between 0.02% and 60% during the stretch and release activity periods, and it is preferably gradually stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr. The tissue specimen is preferably repetitively stretched and released during activity periods of at least 5 seconds in duration, and the activity periods are preferably separated by rest periods of at least five seconds in duration. The membrane preferably has a pair of spaced, upwardly extending support walls thereon, and the support walls are preferably made of a biocompatible material and they have opposed support surfaces thereon. The support walls are preferably made of stainless steel screening so that the support surfaces thereof have sufficiently roughened textures to permit attachment of the cells thereto. In this regard, the tissue specimen is attached or allowed to attach to the support surfaces of the support walls, and thereafter the tissue specimen is mechanically stimulated by stretching the membrane to move the support walls apart in accordance with the method.
The apparatus of the instant invention which is operative for performing the above method comprises a resiliently expandable support means which is operative for supporting a tissue specimen and adapted as needed to permit attachment of the tissue specimen thereto. The apparatus further comprises means for expanding the support means to stretch the tissue specimen and control means for controlling the means for expanding the support means to gradually and substantially continuously expand the support means over a prolonged period of time and to repetitively expand or stretch and release the support means over the same period of time. The control means is preferably adapted to control the means for expanding the support means to effect stretching activity in accordance with the method as hereinabove set forth. In a first embodiment of the apparatus, the support means comprises a substantially circular, resiliently flexible membrane having an upwardly facing side and a downwardly facing side, and the tissue specimen is applied to the upwardly facing side of the membrane. In this embodiment, the mechanical manipulating means is engageable with the downwardly facing side of the membrane to deform the membrane upwardly. The support means preferably further comprises a substantially circular support ring on the membrane which extends upwardly from the membrane and has an inwardly facing support surface thereon. The support ring is made of a biocompatible material, such as stainless steel screening, and it has a sufficiently roughened surface texture to permit attachment of the tissue specimen thereto. In a second embodiment of the apparatus, the support means comprises a resiliently flexible membrane having opposite first and second ends and having an upwardly facing side and a downwardly facing side, and the mechanical manipulating means is operative for stretching the cells by separating the first and second ends. In this embodiment, the support means preferably further comprises first and second spaced support walls which extend upwardly from the upwardly facing side of the membrane and have opposed support surfaces thereon. The support walls are also preferably made of a biocompatible screening material, such as stainless steel screening, so that the support surfaces have sufficiently roughened textures to permit attachment of the tissue specimen thereto.
Accordingly, it is a primary object of the instant invention to provide an effective method of growing a tissue specimen in vitro so that the tissue specimen more closely resembles tissue which is grown in vivo.
Another object of the instant invention is to provide a method of mechanically stimulating the growth of a tissue specimen by gradually and substantially continuously stretching the tissue specimen over a prolonged period of time while simultaneously repetitively stretching and releasing the tissue specimen.
Another object of the instant invention is to provide an apparatus for mechanically stimulating the growth of a tissue specimen.
An even further object of the instant invention is to provide an effective apparatus for mechanically stimulating the growth of a tissue specimen by simultaneously gradually stretching the tissue specimen and periodically repetitively stretching and releasing the tissue specimen.
Other objects, features and advantages of the invention shall become apparent as the description thereof proceeds when considered in connection with the accompanying illustrative drawings.